The invention relates to essential fungal genes and their use in identifying antifungal agents.
Fungal infections (mycoses) may be cutaneous, subcutaneous, or systemic. Superficial mycoses include tinea capitis, tinea corporis, tinea pedis, perionychomycosis, pityriasis versicolor, oral thrush, and other candidoses such as vaginal, respiratory tract, biliary, eosophageal, and urinary tract candidoses. Systemic mycoses include systemic and mucocutaneous candidosis, cryptococcosis, aspergillosis, mucormycosis (phycomycosis), paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, and sporotrichosis. Fungal infections can also contribute to meningitis and pulmonary or respiratory tract diseases. Opportunistic fungal infections proliferate, especially in patients afflicted with AIDS or other diseases that compromise the immune system.
Examples of pathogenic fungi include dermatophytes (e.g., Microsporum canis and other M. spp.; and Trichophyton spp. such as T. rubrum, and T. mentagrophytes), yeasts (e.g., Candida albicans, C. Tropicalis, or other Candida species), Torulopsis glabrata, Epidermophyton floccosum, Malassezia furfur (Pityropsporon orbiculare, or P. ovale), Cryptococcus neoformans, Aspergillus fumigatus, and other Aspergillus sp., Zygomycetes (e.g., Rhizopus, Mucor), Paracoccidioides brasiliensis, Blastomyces dermatitides, Histoplasma capsulatum, Coccidioides immitis, and Sporothrix schenckii. 
Various strains of the fungus Aspergillus sp. cause aspergillosis, a potentially life-threatening disease in humans and other mammals. The clinical manifestations of aspergillosis in humans are very similar to those observed in rodents and cows. For example, necrosis, angioinvasion, and hematogenous dissemination are common features of aspergillosis in rodent and bovine model systems and in humans. In humans, aspergillosis typically is caused by inhalation of conidia (i.e., asexual spores produced by the fungus). In cattle, pathogenic Aspergillus typically enter the animal through the forestomach and then disseminate through the blood of the animal. Putative virulence factors produced by pathogenic species of Aspergillus include hydroxymate siderophores (i.e., compounds that compete with human iron-binding proteins to acquire iron to support fungal growth), lipids having the ability to inhibit complement and phagocytosis, and proteinases that can degrade elastin and other substrates.
The invention is based on the discovery of four new genes in the fungus Aspergillus nidulans that are essential for survival. These genes are referred to herein as AN97, AN80, AN17, and AN85; for convenience, the polypeptides encoded by these genes are referred to herein as xe2x80x9cAN polypeptides.xe2x80x9d The genes encoding the AN polypeptides are useful molecular tools for identifying similar genes in pathogenic microrganisms, such as pathogenic strains of Aspergillus (e.g. Aspergillus fumigatus and Aspergillus flavus). In addition, the AN polypeptides and the essential genes encoding them are useful targets for identifying compounds that are inhibitors of the pathogens in which the AN polypeptides are expressed. Such inhibitors inhibit fungal growth by being fungistatic (e.g., inhibiting reproduction or cell division) or by being fungicidal (i.e., by causing cell death).
The invention, therefore, features an isolated AN97 polypeptide having the amino acid sequence set forth as partial sequences in SEQ ID NOs 2 and 29, or conservative variations thereof. Nucleic acids encoding AN97 also are included within the invention. In particular, the invention includes an isolated nucleic acid of (a) SEQ ID NO:1, as depicted in FIG. 1, or degenerate variants thereof; (b) SEQ ID NO:1, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and that hybridize under stringent conditions to genomic DNA encoding the polypeptide as partial sequences in SEQ ID NOs 2 and 29.
The invention also features an isolated AN80 polypeptide having the amino acid sequence set forth in SEQ ID NO:5, or conservative variations thereof. Nucleic acids encoding AN80 also are included. In particular, the invention includes an isolated nucleic acid of: (a) SEQ ID NO:4, as depicted in FIG. 2, or degenerate variants thereof; (b) SEQ ID NO:4, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and which hybridize under stringent conditions to genomic DNA encoding the polypeptide of SEQ ID NO:5.
The invention also includes an isolated AN85 polypeptide having the amino acid sequence set forth as partial sequences in SEQ ID NOs:8, 30, 31, and 32, or conservative variations thereof. Nucleic acids encoding AN85 also are included. In particular, the invention includes an isolated nucleic acid of: (a) SEQ ID NO:7, as depicted in FIG. 3, or degenerate variants thereof; (b) SEQ ID NO:7, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and which hybridize under stringent conditions to genomic DNA encoding the polypeptide set forth as partial sequences in SEQ ID NOs:8, 30, 31, and 32.
The invention also features an isolated AN17 polypeptide having the amino acid sequence set forth as partial sequences in SEQ ID NOs:11, 33, 34, and 35, or conservative variations thereof. Nucleic acids encoding AN17 also are included. In particular, the invention includes an isolated nucleic acid of: (a) SEQ ID NO:10, as depicted in FIG. 4, or degenerate variants thereof; (b) SEQ ID NO:10, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and which hybridize under stringent conditions to genomic DNA encoding the polypeptide set forth as partial sequences in SEQ ID NOs:11, 33, 34, and 35.
The invention also includes isolated nucleic acids that are at least 15 base pairs in length and which hybridize under stringent conditions to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, and SEQ ID NO:10. In addition, the invention includes allelic variants (i.e., genes encoding isozymes) of the genes encoding AN97, AN17, AN80, and AN85. For example, the invention includes genes that encode an AN polypeptide but which gene includes point mutation, deletion, promoter variant, or splice site variant, provided that the resulting AN polypeptide functions as an AN polypeptide (e.g., as determined in a complementation assay, as described herein and elsewhere). Also included within the invention are isolated nucleic acid molecules containing the cDNA sequences contained with ATCC accession numbers 209473, 209472, 209484, and 209471(deposited with the American Type Culture Collection, 10801 University Boulevard, Manasses, Va. 20110-2209 on Nov. 19, 1997), as well as polypeptides encoded by the cDNA sequences of these nucleic acid molecules. encoded by the cDNA sequences of these nucleic acid molecules.
Identification of the AN97, AN17, AN80, and AN85 genes and the determination that they are essential allows homologs of these genes to be found in other organisms (e.g., fungi, such as yeast like S. cerevisiae; mammalian cells, such as human or murine cells; or plant cells). Thus, the AN polypeptides used not only can be as a model for identifying similar essential genes in other Aspergillus strains, but also to identify homologous essential genes in other organisms, e.g., S. cerevisiae. Because such genes are homologs, they can be expected to be essential for survival without the need for extensive characterization of the homologous gene or polypeptide. Even though some such homologous genes may have previously been identified, the invention allows one to determine that such genes are essential for survival. Having identified such homologous genes as essential, these genes and the polypeptides encoded by these genes can be used to identify compounds that inhibit the growth of the host organism (e.g., compounds that are fungicidal or fungistatic against pathogenic strains of the organism).
As used herein, the term xe2x80x9cyeastxe2x80x9d refers to organisms of the order Saccharomycetales, which includes yeast such as Saccharomyces and Candida. As described below, several homologs of the AN polypeptides have been identified in the yeast S. cerevisiae and are essential for survival. Given the identification of such genes as essential in S. cerevisiae, homologs of these essential yeast genes can also be found in pathogenic yeast strains (e.g., Candida albicans). The S. cerevisiae polypeptide and gene termed D9798.4 are homologs of the AN97 polypeptide and gene. The D9798.4 polypeptide and nucleic acid are depicted in FIG. 5, and are set forth in SEQ ID NOs:14 and 13, respectively (GenBank Accession No. U32517). As described herein, various methods of the invention can utilize the D9798.4 polypeptide or conservative variations thereof. Also useful are isolated nucleic acids of (a) SEQ ID NO:13, as depicted in FIG. 5, or degenerate variants thereof; (b) SEQ ID NO:13, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and which hybridize under stringent conditions to genomic DNA encoding the polypeptide of SEQ ID NO:14.
Yeast homologs of the AN85 and AN80 polypeptides and genes also have been identified as being essential for survival, and these homologs can be used in the methods described herein. As described above for AN97, conservative variations, degenerate variants, complementary sequences, fragments, and nucleic acids in which T is replaced by U also can be used in various methods of the invention. Two homologs of AN85 have been identified. The amino acid and nucleic acid sequences of the AN85 homolog termed YGR010W are depicted in FIG. 6 (GenBank Accession No. Z72795); these sequences are set forth as SEQ ID NOs:17 and 16, respectively. The amino acid and nucleic acid sequences of the AN85 homolog termed L8543.16 are depicted in FIG. 7 (GenBank Accession No. U20618); these sequences are set forth as SEQ ID NOs:20 and 19, respectively. The AN80 polypeptide and gene have a homolog in yeast, termed L8004.2, the amino acid and nucleic acid sequences of which are depicted in FIG. 8 (GenBank Accession No. U53876). These sequences are set forth as SEQ ID NOs:23 and 22, respectively.
The term AN97 polypeptide or gene as used herein is intended to include the polypeptide and gene set forth in FIG. 1 herein, as well as homologs of the sequences set forth in FIG. 1. For example, encompassed by the term AN97 gene are degenerate variants of the nucleic acid sequence set forth in FIG. 1. (SEQ ID NO:1). Degenerate variants of a nucleic acid sequence exist because of the degeneracy of the amino acid code; thus, those sequences that vary from the sequence represented by SEQ ID NO:1, but which nonetheless encode an AN97 polypeptide are included within the invention. Likewise, because of the similarity in the structures of amino acids, conservative variations can be made in the amino acid sequence of the AN97 polypeptide while retaining the function of the polypeptide (e.g., as determined in a complementation assay, as described herein and elsewhere). AN97 polypeptides and genes identified in additional Aspergillus strains may be such conservative variations or degenerate variants of the particular AN97 polypeptide and nucleic acid set forth in FIG. 1 (SEQ ID NOs:2 and 29; and 1, respectively). The AN97 polypeptide and gene share at least 80%, e.g., 90%, sequence identity with SEQ ID NOs:2 and 29; and 1, respectively. Regardless of the percent sequence identity between the AN97 sequence and the sequence represented by SEQ ID NOs:1 and 2, the AN97 genes and polypeptides encompassed by the invention are able to complement for the lack of AN97 function (e.g., in a temperature-sensitive mutant) in a standard complementation assay. AN97 genes that are identified and cloned from additional Aspergillus strains, and pathogenic strains in particular, can be used to produce AN97 polypeptides for use in the various methods described herein, e.g., for identifying antifungal agents. Likewise, the term AN80 encompasses homologues and conservative and degenerate variants of the sequences depicted in FIG. 2. Such homologues, conservative variations, and degenerate variants of AN17, AN85, and AN80 also are included within the invention. Excluded from the invention are the naturally-occurring homologs of AN polypeptides and nucleic acids found in S. cerevisiae (D9798.4, L8543.16, YGR010W, and L8004.2), although methods employing such polypeptides and nucleic acids are encompassed by the invention.
The AN97, AN17, AN80, and AN85 genes have been identified and shown to be essential for survival, these AN polypeptides and their yeast homologs (e.g., D9798.4, L8543.16, YGR010W, and L8004.2) can be used to identify antifungal agents. More specifically, these AN polypeptides and their yeast homologs can be used, separately or together, in assays to identify test compounds which bind these polypeptides. Such test compounds are expected to be antifungal agents, in contrast to compounds that do not bind AN97, AN17, AN80, AN85, D9798.4, L8543.16, YGR010W, and/or L8004.2. As described herein, any of a variety of art-known methods can be used to assay for binding of test compounds to the polypeptides. The invention includes, for example, a method for identifying an antifungal or anti-yeast agent where the method entails: (a) contacting an AN polypeptide, or homolog thereof, with a test compound; (b) detecting binding of the test compound to the AN polypeptide or homolog; and (c) determining whether a test compound that binds the AN polypeptide or homolog inhibits growth of fungi or yeast, relative to growth of fungi or yeast cultured in the absence of the test compound that binds the AN polypeptide or homolog, as an indication that the test compound is an antifungal or anti-yeast agent.
In various embodiments, the AN polypeptide is derived from a non-pathogenic or pathogenic Aspergillus strain, such as Aspergillus nidulans, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger. Preferably, homologs thereof are derived from the yeast Saccharomyces cerevisiae. The test compound can be immobilized on a substrate, and binding of the test compound to the AN polypeptide or homolog can be detected as immobilization of the AN polypeptide or homolog on the immobilized test compound, e.g., in an immunoassay with an antibody that specifically binds AN97.
If desired, the test compound can be a test polypeptide (e.g., a polypeptide having a random or predetermined amino acid sequence; or a naturally-occurring or synthetic polypeptide). Alternatively, the test compound can be a nucleic acid, such as a DNA or RNA molecule. In addition, small organic molecules can be tested. The test compound can be a naturally-occurring compound or it can be synthetically produced, if desired. Synthetic libraries, chemical libraries, and the like can be screened to identify compounds that bind the AN polypeptides. More generally, binding of test compound to the AN polypeptide or homolog can be detected either in vitro or in vivo. Regardless of the source of the test compound, the AN polypeptides described herein can be used to identify compounds that are fungicidal or fungistatic to a variety of pathogenic or non-pathogenic strains.
In an exemplary method, binding of a test compound to an AN polypeptide can be detected in a conventional two-hybrid system for detecting protein/protein interactions (e.g., in yeast or mammalian cells). Generally, in such a method, (a) the AN polypeptide is provided as a fusion protein that includes the AN polypeptide fused to (i) a transcription activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor; (b) the test polypeptide is provided as a fusion protein that includes the test polypeptide fused to (i) a transcription activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor; and (c) binding of the test polypeptide to the AN polypeptide polypeptide is detected as reconstitution of a transcription factor. The yeast homologs can be used in similar methods. Reconstitution of the transcription factor can be detected, for example, by detecting transcription of a gene that is operably linked to a DNA sequence bound by the DNA-binding domain of the reconstituted transcription factor (See, for example, White, 1996, Proc. Natl. Acad. Sci. 93:10001-10003 and references cited therein and Vidal et al., 1996, Proc. Natl. Acad. Sci. 93:10315-10320).
In an alternative method, an isolated nucleic acid molecule encoding an AN polypeptides is used to identify a compound that decreases the expression of the AN polypeptide in vivo. Such compounds can be used as antifungal agents. To discover such compounds, cells that express an AN polypeptide are cultured, exposed to a test compound (or a mixture of test compounds), and the level of expression or activity is compared with the level of AN polypeptide expression or activity in cells that are otherwise identical but that have not been exposed to the test compound(s). Many standard quantitative assays of gene expression can be utilized in this aspect of the invention.
In order to identify compounds that modulate expression of an AN polypeptide (or homologous sequence), the test compound(s) can be added at varying concentrations to the culture medium of cells that express an AN polypeptide (or homolog), as described above. Such test compounds can include small molecules (typically, non-protein, non-polysaccharide chemical entities), polypeptides, and nucleic acids. The expression of the AN polypeptide is then measured, for example, by Northern blot PCR analysis or RNAse protection analyses using a nucleic acid molecule of the invention as a probe. The level of expression in the presence of the test molecule, compared with the level of expression in its absence, will indicate whether or not the test molecule alters the expression of the AN polypeptide. Because the AN polypeptides are essential for survival, test compounds that inhibit the expression and/or function of the AN polypeptide will inhibit growth of the cells or kill the cells.
Compounds that modulate the expression of the polypeptides of the invention can be identified by carrying out the assay described above and then measuring the levels of the AN polypeptides expressed in the cells, e.g., by performing a Western blot analysis using antibodies that bind an AN polypeptide.
The invention further features methods of identifying from a large group of mutants those strains that have conditional lethal mutations. In general, the gene and corresponding gene product are subsequently identified, although the strains themselves can be used in screening or diagnostic assays. The mechanism(s) of action for the identified genes and gene products provide a rational basis for the design of anti-fungal therapeutic agents. These antifungal agents reduce the action of the gene product in a wild type strain, and therefore are useful in treating a subject with that type, or a similarly susceptible type of infection by administering the agent to the subject in a pharmaceutically effective amount. Reduction in the action of the gene product includes competitive inhibition of the gene product for the active site of an enzyme or receptor; non-competitive inhibition; disrupting an intracellular cascade path which requires the gene product; binding to the gene product itself, before or after post-translational processing; and acting as a gene product mimetic, thereby down-regulating the activity. Therapeutic agents include monoclonal antibodies raised against the gene product.
Furthermore, the presence of the gene sequence in certain cells (e.g., a pathogenic fungus of the same genus or similar species), and the absence or divergence of the sequence in host cells can be determined, if desired. Therapeutic agents directed toward genes or gene products that are not present in the host have several advantages, including fewer side effects, and lower overall dosage.
The invention includes pharmaceutical formulations that include a pharmaceutically acceptable excipient and an antifungal agent identified using the methods described herein. In particular, the invention includes pharmaceutical formulations that contain antifungal agents that inhibit the growth of, or kill, pathogenic Aspergillus strains. Such pharmaceutical formulations can be used for treating an Aspergillus infection in an organism. Such a method entails administering to the organism a therapeutically effective amount of the pharmaceutical formulation. In particular, such pharmaceutical formulations can be used to treat aspergillosis in mammals such as humans and domesticated mammals (e.g., cows and pigs). The efficacy of such antifungal agents in humans can be estimated in an animal model system well known to those of skill in the art (e.g., bovine and rodent (e.g., mouse) model systems). These formulations also can be used to treat fungal infections in plants, e.g., by topically applying the antifungal agent to the plant. Alternatively, where the antifungal agent is a polypeptide or an antisense RNA, a gene encoding the polypeptide or expressing the antisense RNA can be transfected into the plant, using conventional techniques, and the polypeptide or antisense RNA can be expressed in the plant.
Also included within the invention are polyclonal and monoclonal antibodies that specifically bind AN97, AN17, AN80, or AN85 polypeptide. Such antibodies can facilitate detection of AN polypeptides in various Aspergillus strains. These antibodies also are useful for detecting binding of a test compound to AN97, AN17, AN80, or AN85 polypeptides (e.g., using the assays described herein). In addition, monoclonal antibodies that bind AN97, AN17, AN80, or AN85 polypeptide are themselves adequate antifungal agents when administered to a mammal, as such monoclonal antibodies are expected to impede one or more functions of AN97, AN17, AN80, or AN85 polypeptide.
As used herein, xe2x80x9cnucleic acidsxe2x80x9d encompass both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid may be double-stranded or single-stranded. Where single-stranded, the nucleic acid may be a sense strand or an antisense strand. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5xe2x80x2 end and one on the 3xe2x80x2 end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5xe2x80x2 non-coding (e.g., promoter) sequences that are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence. The term xe2x80x9cisolatedxe2x80x9d can refer to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an xe2x80x9cisolated nucleic acid fragmentxe2x80x9d is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.
A nucleic acid sequence that is xe2x80x9csubstantially identicalxe2x80x9d to an AN97, AN17, AN80, or AN85 nucleotide sequence is at least 80% or 85% identical to the nucleotide sequence of the Aspergillus AN97, AN80, AN85, and AN17 nucleic acids of SEQ ID NO:1, NO:4, NO:7, and NO:10, respectively, as depicted in FIGS. 1, 2, 3, and 4, respectively. For purposes of comparison of nucleic acids, the length of the reference nucleic acid sequence will generally be at least 40 nucleotides, e.g., at least 60 nucleotides or more nucleotides. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
The AN polypeptides of the invention include, but are not limited to, recombinant polypeptides and natural polypeptides. The invention also encompasses nucleic acid sequences that encode forms of AN97, AN17, AN80, or AN85 polypeptides in which naturally occurring amino acid sequences are altered or deleted. Preferred nucleic acids encode polypeptides that are soluble under normal physiological conditions. Also within the invention are nucleic acids encoding fusion proteins in which a portion of AN97, AN17, AN80, or AN85 is fused to an unrelated polypeptide (e.g., a marker polypeptide or a fusion partner) to create a fusion protein. For example, the polypeptide can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed polypeptides, or to a hemagglutinin tag to facilitate purification of polypeptides expressed in eukaryotic cells. The invention also includes isolated, for example, polypeptides (and the nucleic acids that encode these polypeptides) that include a first portion and a second portion; the first portion includes, e.g., an AN polypeptide, and the second portion includes an immunoglobulin constant (Fc) region or a detectable marker.
The fusion partner can be, for example, a polypeptide which facilitates secretion, e.g., a secretory sequence. Such a fused polypeptide is typically referred to as a preprotein. The secretory sequence can be cleaved by the host cell to form the mature protein. Also within the invention are nucleic acids that encode AN97, AN17, AN80, or AN85 fused to a polypeptide sequence to produce an inactive preprotein. Preproteins can be converted into the active form of the protein by removal of the inactivating sequence.
The invention also includes nucleic acids that hybridize, e.g., under stringent hybridization conditions (as defined herein) to all or a portion of the nucleotide sequence of SEQ ID NO:1, NO:4, NO:7, or NO:10, or their complements. The hybridizing portion of the hybridizing nucleic acids is typically at least 15 (e.g., 20, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 95%, or at least 98%, identical to the sequence of a portion or all of a nucleic acid encoding an AN97, AN17, AN80, or AN85 polypeptide. Hybridizing nucleic acids of the type described herein can be used as a cloning probe, a primer (e.g., a PCR primer), or a diagnostic probe. Nucleic acids that hybridize to the nucleotide sequences of SEQ ID NO:1, NO:4, NO:7, or NO:10 are considered xe2x80x9cantisense oligonucleotides.xe2x80x9d Also included within the invention are ribozymes that inhibit the function of AN97, AN17, AN80, or AN85, as determined, for example, in a complementation assay.
In another embodiment, the invention features cells, e.g., transformed host cells, that contain a nucleic acid is encompassed by the invention. A xe2x80x9ctransformed cellxe2x80x9d is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid encoding an AN polypeptide. Both prokaryotic and eukaryotic cells are included, e.g., bacteria, Aspergillus, yeast, and the like.
The invention also features genetic constructs (e.g., vectors and plasmids) that include a nucleic acid of the invention which is operably linked to a transcription and/or translation sequence to enable expression, e.g., expression vectors. By xe2x80x9coperably linkedxe2x80x9d is meant that a selected nucleic acid, e.g., a DNA molecule encoding an AN polypeptide, is positioned adjacent to one or more sequence elements, e.g., a promoter, which directs transcription and/or translation of the sequence such that the sequence elements can control transcription and/or translation of the selected nucleic acid.
The invention also features purified or isolated AN97, AN17, AN80, and AN85 polypeptides. As used herein, both xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d mean any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Thus, the terms xe2x80x9cAN97 polypeptidexe2x80x9d (or AN97), xe2x80x9cAN17 polypeptidexe2x80x9d (or AN17), xe2x80x9cAN80 polypeptidexe2x80x9d (or AN80), or xe2x80x9cAN85 polypeptidexe2x80x9d (or AN85) include full-length, naturally occurring AN97, AN17, AN80, or AN85 proteins, respectively, as well as recombinantly or synthetically produced polypeptides that correspond to a full-length, naturally occurring AN97, AN17, AN80, or AN85 protein, or to a portion of a naturally occurring or synthetic AN97, AN17, AN80, or AN85 polypeptide.
A xe2x80x9cpurifiedxe2x80x9d or xe2x80x9cisolatedxe2x80x9d compound is a composition that is at least 60% by weight the compound of interest, e.g., an AN97 polypeptide or antibody. Preferably the preparation is at least 75% (e.g., at least 90% or 99%) by weight the compound of interest. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Preferred AN97, AN17, AN80, AN85 polypeptides include a sequence substantially identical to all or a portion of a naturally occurring AN97, AN17, AN80, or AN85 polypeptide, e.g., including all or a portion of the sequences shown in FIGS. 1, 2, 3, and 4, respectively. Polypeptides xe2x80x9csubstantially identicalxe2x80x9d to the AN polypeptide sequences described herein have an amino acid sequence that is at least 80% or 85% (e.g., 90%, 95% or 99%) identical to the amino acid sequence of the AN97, AN80, AN85 or AN17 polypeptides of SEQ ID NOs:2 and 29; NO:5; NOs:8, 30, 31, and 32; and NOs:11, 33, 34, and 35, respectively. For purposes of comparison, the length of the reference AN polypeptide sequence will generally be at least 16 amino acids, e.g., at least 20 or 25 amino acids.
In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Where a particular polypeptide is said to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference polypeptide. Thus, a polypeptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It also might be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, other polypeptides also will meet the same criteria.
The invention also features purified or isolated antibodies that specifically bind to an AN polypeptide. By xe2x80x9cspecifically bindsxe2x80x9d is meant that an antibody recognizes and binds a particular antigen, e.g., an AN97, AN17 polypeptide, but does not substantially recognize and bind other molecules in a sample, e.g., a biological sample that naturally includes AN97, AN17, AN80, or AN85. In one embodiment the antibody is a monoclonal antibody.
In another aspect, the invention features a method for detecting an AN polypeptide in a sample. This method includes: obtaining a sample suspected of containing AN97, AN17, AN85, or AN80; contacting the sample with an antibody that specifically binds an AN97, AN17, AN85 or AN80 polypeptide under conditions that allow the formation of complexes of an antibody and AN97, AN17, AN85 or AN80; and detecting the complexes, if any, as an indication of the presence of AN97, AN17, AN85 or AN80 in the sample.
Also encompassed by the invention is a method of obtaining a gene related to (i.e., a functional homologue of) the AN97, AN17, AN85, or AN80 gene. Such a method entails obtaining a labeled probe that includes an isolated nucleic acid which encodes all or a portion of AN97, AN17, AN85, or AN80, or a homolog thereof (e.g., D9798.4, L8543.16, YGR010W, or L8004.2); screening a nucleic acid fragment library with the labeled probe under conditions that allow hybridization of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes; isolating labeled duplexes, if any; and preparing a full-length gene sequence from the nucleic acid fragments in any labeled duplex to obtain a gene related to the AN97, AN17, AN85, or AN80 gene.
The invention offers several advantages. By combining gene knockout assays, as described herein, with assays of conditional sensitivity, we have identified genes that are truly essential, i.e., genes whose absence is fungicidal to Aspergillus. In addition, the methods for identifying antifungal agents can be configured for high throughput screening of numerous candidate antifungal agents.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the case of a conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative and are not intended to limit the scope of the invention, which is defined by the claims.